Due to delayed sample processing, more alterations were detected in the samples collected in K3-EDTA than in the samples collected in Na-heparin. eleven parameters remained stable despite delayed sample handling. Due to delayed sample processing, more alterations were detected in the samples collected in K3-EDTA than in the samples collected in Na-heparin. The type of CD11b clone influenced the reduction of fluorescence intensity only in samples collected in K3-EDTA, where the alterations were contrary to the changes observed in Na-heparin. Conclusions Delayed sample processing causes considerable immunohenotypic alterations, which can lead to false interpretation of the results. If delayed sample evaluation is unavoidable, markers that remain more stable over time should be considered with more excess weight in the diagnosis of MDS. Key words: myelodysplastic syndromes, circulation cytometry, pre-analytical error Introduction Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell disorders, therefore no single specific marker or method exists for diagnosing all MDS cases. At the same time, MDS are characterized by variable clinical end result, which makes precise diagnosis and classification of cases by prognostic category vital. The algorithm currently applied for the diagnosis and prognostic classification of MDS considers several parameters from morphologic and cytogenetic examinations, and in addition, it also recommends the application of other methods, such as circulation cytometry (FCM) (immunophenotype and its alterations on day 1 and day 2 in samples collected into K3-EDTA (N = 23) or Na-heparin (N = 16). Samples were kept on room heat prior to analysis. Table 1 TNFRSF9 Clinical and laboratory parameters of patients B12, folic acid concentrations) as well as morphological, cytogenetic, and circulation cytometric examination. WBC, Hb, Plt and ANC parameters were measured in peripheral blood samples of patients with suspected MDS or MPN. Open in a separate window In the second group residual peripheral blood (PB) samples of eight patients with no haematological malignancy were collected in one tube K3-EDTA and one tube Na-heparin for circulation cytometry measurements, and they were Glutathione examined with different clones of CD11b monoclonal antibodies. We conducted our studies in compliance with the principles of the Declaration of Helsinki. Informed Glutathione consent was obtained from each participant. The Hungarian Medical Research Council granted permission for our studies (20582-2/2017/EKU). Methods Bone marrow samples were analysed for MDS by eight-colour labelling. The antibodies and clones we examined are shown in Table 2. CD14, CD11b, HLA-DR, CD45, CD64, CD13, CD15, CD34, CD71, CD117, CD300e, CD4, and CD10 markers were purchased from Becton Dickinson Biosciences (San Jose, USA); CD33, CD16, and CD13 markers were purchased from Beckman Coulter, (Brea, USA); CD45 marker was purchased from Invitrogen (Thermo Scientific Inc., Walthman, USA); and HLA-DR marker was purchased from Biolegend (San Diego, USA). Antibody combinations were added to 50 mL BM or PB samples (1 x 106 cells) and incubated for 15 minutes in the dark at room heat. Then 1 mL lysing answer was added to each tube and samples were incubated for an additional 8 moments. Finally, samples were washed Glutathione once in phosphate-buffered saline (PBS) and suspended in 500 mL Glutathione 1% paraformaldehyde (PFA). The FACS Canto II circulation cytometer (Becton Dickinson Biosciences, San Glutathione Jose, USA) was utilized for cell analysis. To make the results comparable, the circulation cytometer was calibrated daily, using Cytometer Setup and Tracking fluorescent microbeads (Cat No. 641319, Becton Dickinson Biosciences, San Jose, USA) and Autocomp software as recommended by the manufacturer. Data were analysed by FACS Diva version 6.1.3 (Becton Dickinson Biosciences, San Jose, CA, USA) and Kaluza Softwares version 1.2 (Beckman Coulter, Brea, CA, USA). Table 2 Antibody combinations used in circulation cytometric examination for the diagnosis of MDS = day 0, day 1 and day 2), of which mean fluorescence intensity (MFI) values, strong.